Page 149 - 2020_01-Haematologica-web

The vascular niche in chronic myeloid leukemia
1271 and imatinib than when they had been treated with vehicle or imatinib alone. Short-term homing of CML-ini- tiating cells to BM (P=0.03) (Figure 1F) and spleen (P=0.048) (Figure 1F) of GMI-1271-treated recipients was impaired compared to that of vehicle-treated recipients. Long-term engraftment of CML-initiating clones, as mea- sured by the enumeration of distinct proviral integration events in splenic tissue of diseased mice by Southern blot- ting, was significantly reduced in primary mice treated with imatinib plus GMI-1271 compared to those treated with imatinib alone (P=0.007) (Figure 1G, H).
In summary, these data suggest that in comparison to monotherapy with imatinib, inhibition of E-selectin in combination with imatinib mildly prolongs survival in mice with a CML-like myeloproliferative neoplasia via a reduction of homing and long-term engraftment of CML- initiating clones. Inhibition of E-selectin also reduces the number of human CML cells adhering to E-selectin.
Inhibition of E-selectin leads to an increase of Scl/Tal1 in BCR-ABL1+ leukemia-initiating cells
In order to explain the prolonged survival of mice trea- ted with imatinib and GMI-1271, we tested the adhesion and gene expression of cell cycle-relevant genes and tran- scription factors in LIC in the presence of GMI-1271. To do so, we plated BCR-ABL1+ Lin- c-Kit+ BM cells from mice with CML on E-selectin-coated plates in the presence of vehicle, GMI-1271,22 imatinib23,24 or the combination of GMI-1271 plus imatinib (Figure 2A). As expected, this revealed that treatment with GMI-1271 (P=0.02) or GMI- 1271 plus imatinib (P=0.029) significantly reduced the number of adherent cells (Figure 2B). In a competitive inhi- bition assay we plated BCR-ABL1+ BaF3 cells, which were used because sufficient numbers of LIC can only be retrieved from a large number of diseased mice, on plates pre-coated with E-selectin, while adding soluble E-selectin
AB
to the BCR-ABL1+ BaF3 cells treated with GMI-1271. This reversed the decreased adhesion of leukemia cells in the presence of GMI-1271 (P=0.021) (Online Supplementary Figure S3A) suggesting a specific role of E-selectin in this process. Furthermore, non-adhesion of primary murine BCR-ABL1+ LIC (P=0.017) (Figure 2C) or K562 cells (P=0.007) (Online Supplementary Figure S3B), which were tested in order to show the validity also in a human cell line, to E-selectin in the presence of GMI-1271 led to an increase of the transcription factor and cell cycle regula- tor25 stem cell leukemia/T-cell acute lymphocytic leukemia protein 1 (Scl/Tal1). Other transcription factors, however, did not significantly change in K562 cells (Online Supplementary Figure S3C-G, Online Supplementary Table S1). Conversely, addition of soluble E-selectin to BCR- ABL1+ BaF3 cells plated on E-selectin in the presence of GMI-1271 (as in Online Supplementary Figure S3A) decreased the expression of Scl/Tal1 (P=0.028) (Online Supplementary Figure S4A). In summary, these data suggest that E-selectin is involved in adhesion of CML cells and that non-adhesion leads to increased expression of Scl/Tal1.
Scl/Tal1 regulates CD44 expression
As SCL/TAL1 is a known transcriptional regulator involved in various hematologic malignancies and cell cycle progression,25 we performed a gene expression screen after knockdown of SCL/TAL1 in K562 cells (data deposited in the GEO-Expression database, GSE92251). This revealed that knockdown of SCL/TAL1 led to an upregulation of CD4426 (Figure 3A). As we had previously shown CD448 and its receptor E-selectin6,7 to be important mediators of the engraftment of LIC in CML, we hypoth- esized that SCL/TAL1 may regulate the expression of CD44 on CML cells, where CD44 is known to be overex- pressed8 – albeit through an unknown mechanism.
C
Figure 2. Inhibition of E-selectin leads to an increase of Scl/Tal1 in BCR-ABL1+ leukemia-initiating cells. (A) Schematic of an in vitro adhesion assay, in which 20,000 GFP+ (BCR-ABL1+) Lin- c-Kit+ bone marrow cells from mice with chronic myeloid leukemia treated with vehicle, GMI-1271, imatinib or the combination of GMI-1271 plus imatinib were plated on recombinant E-selectin in the presence of the respective drugs for 6 h. (B) Percentage of adherent GFP+ (BCR-ABL1+) Lin- c-Kit+ of total cells after 6 h of incubation on recombinant E-selectin in the presence of vehicle (black), 20 mM GMI-1271 (dark gray), 10 mM imatinib (light gray) or imatinib plus GMI-1271 (white) and several washing steps, normalized by the number of live cells. Twenty thousand cells per well were plated in triplicate. The percentage of adherent cells was reduced by treatment with GMI-1271 or imatinib plus GMI-1271 compared to vehicle [P=0.02 or P=0.029, respectively, analysis of variance (ANOVA); Tukey test, n=4]. (C) Relative expression of Scl/Tal1 in all GFP+ (BCR-ABL1+) Lin- c-Kit+ cells plated on recombinant E-selectin as in (A) and (B) in the presence of vehicle (black), GMI-1271 (dark gray), imatinib (light gray) or imatinib plus GMI-1271 (white). The expression of Scl/Tal1 in GMI-1271-treated cells was significantly increased compared to that of vehicle-treated cells (P=0.017, ANOVA; Tukey test, n=3). BM: bone marrow; CML: chronic myeloid leukemia; qPCR: real-time polymerase chain reaction.
haematologica | 2020; 105(1)
139